Wnt/β-catenin信号通路活化的人滑膜细胞对软骨细胞的影响

Effect of Human Synovial Cells with Wnt/β-catenin Signal Pathway Activated on Human Chondrocytes

目的:研究Wnt/β-catenin信号通路活化的人滑膜细胞对软骨细胞的作用探讨骨关节炎(OA)发生及进展的可能途径和分子机制。方法:β-catenin过表达慢病毒载体转染正常人滑膜细胞,荧光显微镜观察病毒转染情况.Western blot方法:检测滑膜细胞核内β-catenin蛋白表达。Transwell小室共培养人滑膜细胞与正常人软骨细胞,倒置显微镜观察滑膜细胞对软骨细胞生长形态变化的影响。酶联免疫吸附(ELISA)法检测不同时间点共培养体系中软骨细胞培养上清液中MMP-7的变化找出MMP-7变化最明显的时间点。同时在最佳时间点ELISA方法:检测软骨细胞培养上清液CTX-Ⅱ、COMP的水平。结果:慢病毒转染滑膜细胞后茨光显微镜下可见多数滑膜细胞表达荧光.Western blot检测发现滑膜细胞胞浆及胞核中β-catenin表达明显上调(P<0.01)。通路活化的人滑膜细胞与软骨细胞共培养后,光镜下观察软骨细胞生长形态变化不明显,但随共培养时间的延长软骨细胞生长受到抑制胞体边缘发白壁强度降低。ELISA结果:显示通路活化的人滑膜细胞与软骨细胞共培养后软骨细胞上清液中MMP-7表达明显升高,与正常组相比有显著差异(P<0.01)软骨细胞上清液MMP-7的变化有一定的时间依赖性在共培养2h、6h、12h时逐渐升高;24h时略有降低且在共培养6h时变化最为明显。而共培养6h时通路激活组软骨细胞培养上清液中COMP、CTX-Ⅱ水平与正常对照组相比均明显升高(P<0.01)。结论:β-catenin过表达慢病毒转染正常人滑膜细胞可成功激活Wnt/β-catenin信号通路,Wnt/β-catenin信号通路活化的滑膜细胞可影响正常软骨细胞的生长,并引起滑膜-软骨体系内环境异常,促使软骨基质的降解这可能是滑膜炎促进OA发生、发展的机制之一。

Objective：This study was designed to research the contributions of synovial cells whose Wnt/β-catenin signal pathway are activated to the cartilage cells,to discuss the ways and molecular mechanism of Osteoarthritis（OA）.Methods：β-catenin over expressing lentivirus vector was constructed to transfect synovial cells of Human and fluorescence microscope was used to observe viral transfection efficiency.Expression of β-catenin protein of the synovial cells was detected by Western blot.Synovial cells and chondrocytes were cultured in transwell chamber and the influences of synovial cells to chondrocytes were observed under optical inverted microscope.Changes of MMP-7 in culture supernatant of chondrocytes was tested by Enzyme linked immunosorbent assay（ELISA） method at different time points to find the action time that synovial cells influence the chondrocytes best.Moreover,ELISA method was used to test CTX-Ⅱ,COMP levels in the culture supernatant of chondrocytes at optimal time point.Results：After been transfected by lentivirus vector,fluorescent microscopy showed that most of the synovial cells were arise fluorescence.Expression ofβ-catenin increased significantly in the cytoplasm and nucleus of synovial cells（P〈0.01）.The growth morphology of chondrocytes did not change significantly under light microscope after co-cultured with synovial cells which Wnt/β-catenin signal pathway are activated.But chondrocytes’ growth and adhesion force was reduced with the prolongation of the culture time,this may be related to the degradation of extracellular matrix.ELISA results showed that MMP-7 expression of chondrocytes supernatant was significantly increased,there was a large difference compared with the normal group（P〈0.01）.MMP-7 expression of chondrocytes culture supernatant were time-dependent.It increased gradually with the time prolonging at 2 h,6 h and 24 h,but decreased slightly at 24 h.The expression of MMP-7 changed significantly after 6 hours’ co-culture.ELISA results showed that the level of COMP,CTX-Ⅱ in the supernatant of chondrocytes within singal pathway activated group was significantly higher than that in normal control group at 6 hours’ co-culture（P〈0.01）.Conclusions：β-catenin over expressing lentivirus can activate Wnt/β-catenin signaling pathway.The grouth of chondrocytes can be affected by synovial cells whose cause the abnormal of internal environment within synovial-cartilage system,promote cartilage matrix degradation at same time.This may be exist molecular interactions between two cells as a possible mechanism of synovitis promoting OA.